Project Summary The human gut microbiota is a complex assemblage of species that contributes to host health through diverse mechanisms, including nutrient production and increased resistance to pathogen colonization. The composition of the gut community has been linked to numerous human diseases, but much remains to be learned about the factors that govern microbiota composition and stability. A relatively unexplored component of microbiota composition is the role of competitive interactions between bacteria, such as the antagonistic interactions mediated by type VI secretion systems (T6SSs). These protein complexes are found in Gram- negative bacteria and are used by many species, including human gut symbionts, to inject toxins into bacterial competitors. By preventing vulnerable strains from establishing in proximity to T6SSs-possessing aggressors, T6SSs may have a substantial effect on the composition and organization of bacterial communities, as well as the outcomes of opportunistic infections. However, it is challenging to examine these potential functions of the T6SS in vivo, as common animal models, like humans, have highly complex and variable gut communities. These traits pose an obstacle to identifying specific interactions that significantly affect the microbiota or host. The honey bee (Apis mellifera) is an experimentally tractable model organism with a simple and highly conserved gut microbiota comprised of only nine species. Bee gut microbes Snodgrassella alvi and Gilliamella apicola encode T6SSs and are the dominant Gram-negative taxa in the bee small intestine, where they form spatially structured communities. Experimental manipulation of this community provides an opportunity to understand the effect of T6SSs on community structure and composition of gut communities. Additionally, a Serratia marcescens strain isolated from sick bees has proven to be lethal to bees upon oral inoculation. This strain encodes a T6SS similar to the aggressively antibacterial T6SS of Se. marcescens Db10. This isolate will be used to determine the role of T6SSs in mediating interactions between an opportunistic pathogen and the commensal gut community. To elucidate the role of T6SSs in the commensal gut community and during infection, the competitive capabilities of T6SS-deficient Sn. alvi and Se. marcescens mutants will be compared to wild type strains. The toxin proteins secreted through the Sn. alvi T6SSs will be identified through proteomic methods to expand our understanding of T6SS function and toxin diversification. Micrographic methods will be used to examine in vivo physical interactions between bacteria and disturbance of community structure. Finally, to better understand the interaction between the gut community and an invading pathogen, two high- throughput genomics screening methods will be used to identify Se. marcescens genes that are differentially expressed or important for the fitness in the presence of the microbiota. This work will expand our understanding of the effect of T6SS-mediated intercellular competition on a commensal gut microbiota and, ultimately, on host health.